Aqueous fibroin solution and production method thereof, and article configured to include fibroin

Abstract

To provide an aqueous fibroin solution having excellent storage stability and excellent foamability, and a production method thereof.SOLUTION: The aqueous fibroin solution contains fibroin and a compound represented by the general formula (1) or (2) in the figure.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to an aqueous solution of fibroin, a method for producing the same, and an article comprising fibroin. [Background technology]

[0002] Fibroin, the main component of silk thread, is highly biocompatible and has long been used in surgical sutures, and is known for its high safety. Recently, there has been active research into applying various foams such as gels, sponges, films, and nonwoven fabrics prepared from fibroin aqueous solutions to medical fields such as cell culture scaffolds, wound dressings, artificial skin, and artificial bone.

[0003] Conventionally, the method for producing aqueous fibroin solutions generally involves scouring unrefined fibroin raw materials such as cocoons and raw silk, dissolving them in a highly concentrated neutral salt solution, and then desalting them by methods such as dialysis or ultrafiltration. However, the resulting aqueous fibroin solution easily undergoes a change in crystal structure and gels due to external stimuli or changes over time. Therefore, there has been a need for an aqueous fibroin solution with high storage stability that does not undergo a change in crystal structure.

[0004] Various additives have been investigated to improve the storage stability of fibroin aqueous solutions. For example, Patent Documents 1 and 2 propose adding urea or thiourea as protein denaturants. Furthermore, Patent Document 3 proposes improving the storage stability of fibroin aqueous solutions by adding guanidino group-containing compounds such as arginine. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 7-90182 [Patent Document 2] Japanese Patent Publication No. 2008-169171 [Patent Document 3] Japanese Patent Publication No. 2015-140328 Summary of the Invention Problems to be Solved by the Invention

[0006] The above additive has an action of strongly modifying proteins, thereby preventing gelation of the aqueous solution and improving storage stability. On the other hand, when attempting to form various forms such as gels and sponges from the aqueous solution using the above additive, there is a problem that crystallization hardly occurs and it takes a long time to form.

[0007] Therefore, an object of the present invention is to provide a fibroin aqueous solution having excellent storage stability and being easily formed into various forms, and a method for producing the same. Means for Solving the Problems

[0008] The fibroin aqueous solution according to an embodiment of the present invention is characterized by containing fibroin and a compound represented by the following general formula (1) or (2).

Chemical formula

Chemical formula

[0009] The article of the present invention is characterized by comprising a fibroin and a compound represented by the following general formula (1) or (2).

Chemical formula

Chemical formula

[0010] According to the present invention, it is possible to provide a fibroin aqueous solution which is excellent in long-term storage stability and is easily formed into various forms.

Mode for carrying out the invention

[0011] The fibroin used in this embodiment is a fibrous protein derived from organisms classified under the orders Lepidoptera, Hymenoptera, or Araneae, and may be obtained by genetic engineering. From the viewpoint of ease of raw material availability, fibroin derived from silkworm cocoons is preferred.

[0012] The fibroin used in this embodiment can be made from silkworm cocoons, silk threads, processed silk threads (such as silk yarn), and leftover threads from processed silk threads. In other words, the fibroin in this embodiment can be made from silk-derived materials such as silkworm cocoons, silk threads, processed silk threads (such as silk yarn), and leftover threads from processed silk threads. Fibroin can be obtained from these raw materials by removing sericin using a known scouring method. The obtained fibroin can be dissolved in a high-concentration aqueous solution of lithium bromide or calcium chloride, and then desalted by methods such as dialysis using a semipermeable membrane or ultrafiltration to obtain an aqueous solution. The obtained aqueous solution is unstable and will form a gel and solidify if left at room temperature, so it is preferable to store it refrigerated at around 4°C.

[0013] The fibroin used in this embodiment is not particularly limited in terms of its molecular weight, but higher molecular weight fibroin with a molecular weight of 100,000 or more yields better results. Generally, the higher the molecular weight of fibroin, the lower the storage stability of its aqueous solution tends to be, but if the molecular weight is 100,000 or less, sufficient storage stability is often observed even without adding the additive of the present invention. When the molecular weight of fibroin exceeds 100,000, the storage stability decreases, and the addition of an additive becomes necessary for long-term storage. Furthermore, if the molecular weight is 100,000 or less, the mechanical properties of the various foams formed from the aqueous solution become poor, which is undesirable depending on the application of the foam.

[0014] The fibroin used in this embodiment preferably has a molecular weight of 100,000 or more, more preferably 150,000 or more. Furthermore, since the molecular weight of fibroin derived from domestic silkworms is 350,000, the upper limit of the molecular weight of the fibroin used in this embodiment is effectively 350,000.

[0015] The molecular weight of fibroin can be controlled by the temperature and time during scouring. While fibroin derived from domestic silkworms has a molecular weight of around 350,000, a desired molecular weight can be obtained by changing the scouring temperature and time. Generally, the higher the scouring temperature and the longer the scouring time, the lower the molecular weight of the fibroin obtained.

[0016] The fibroin aqueous solution according to this embodiment is characterized by containing the compound represented by the general formula (1) or (2).

[0017] By containing the above compound, an aqueous solution is obtained that has excellent storage stability and can be easily formed into various forms.

[0018] Common protein denaturants such as urea and guanidino group-containing compounds are thought to inhibit gelation by selectively binding to peptide chains and inhibiting the formation of hydrogen bonds between peptide chains. While such compounds are effective in improving the stability of aqueous solutions, using them when creating various foams with aqueous solutions will result in a longer foam formation time because they inhibit hydrogen bond formation.

[0019] On the other hand, although the mechanism of action of the compound according to this embodiment is not clear, it is thought that it forms ion pairs with carboxyl groups derived from glutamic acid and aspartic acid residues, and that the proximity of peptides is suppressed by steric or electrostatic action. Because the aqueous solution is stabilized by such a mechanism, when external stimuli are applied during the formation of various forms, the peptide chains are more likely to form hydrogen bonds with each other, and the formation of various forms is also thought to be facilitated.

[0020] In general formula (1), R 1 From R 4 Each of these independently represents a hydrogen atom, an aliphatic hydrocarbon group having 1 to 8 carbon atoms which may have substituents, or an aryl group or aralkyl group having 6 to 10 carbon atoms which may have substituents, provided that R 1 From R 4 At least one of them is not a hydrogen atom, X- represents an anion. Also, in general formula (2), CN=C and A both form a ring structure, R 5 X represents a hydrogen atom, an aliphatic hydrocarbon group having 1 to 8 carbon atoms which may have substituents, or an aryl group or aralkyl group having 6 to 10 carbon atoms which may have substituents. - This indicates an anion. Compounds with more carbon atoms than those mentioned above exhibit high hydrophobicity, resulting in hydrophobic interactions preferentially acting over ion pair formation with the peptide chain. Therefore, while this provides a stabilizing effect on aqueous solutions, it makes foam formation difficult.

[0021] The aforementioned R 1 , R 2 , R 3 , R 4 or R 5 Examples of aliphatic hydrocarbon groups having 1 to 8 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, n-octyl, and 2-ethylhexyl groups, and these aliphatic hydrocarbon groups may be bonded to each other to form a ring. Examples of substituents include hydroxyl, amino, alkoxycarbonyl, and carbamoyl groups.

[0022] The aforementioned R 1 , R 2 , R 3 , R 4 or R 5 Examples of aryl or aralkyl groups having 6 to 10 carbon atoms include aryl groups such as phenyl, 1-naphthyl, and 2-naphthyl groups, and aralkyl groups substituted with these, such as methyl, ethyl, n-propyl, isopropyl, and n-butyl groups. Examples of substituents include alkyl groups, hydroxyl groups, amino groups, alkoxycarbonyl groups, and carbamoyl groups.

[0023] In the general formula (2) above, both CN=C and A form a ring structure. For example, both CN=C and A can form pyridine or imidazole. The cation portion of general formula (2) is preferably a heterocyclic aromatic compound. Examples include 1-alkylpyridinium cations and 1,3-dialkylimidazolium cations.

[0024] In the general formulas (1) and (2), X - These can be halide ions, hydroxide ions, or carboxylic acid anions. Examples of halide ions include fluoride ions, chloride ions, bromide ions, and iodide ions.

[0025] Examples of carboxylic acid anions include acetate anions, propionate anions, benzoate anions, tartrate anions, and hydrogen tartrate anions.

[0026] The compounds represented by the general formulas (1) and (2) are not particularly limited as long as they have the above substituents, but water-soluble compounds are preferred from the viewpoint of improving storage stability when added to an aqueous fibroin solution. Compounds exhibiting solubility of 0.01% by mass or more in water are preferred, and more preferably compounds with solubility of 0.1% by mass or more are preferred. If the water solubility is less than 0.01% by mass, a sufficient stability improvement effect cannot be obtained.

[0027] The compounds represented by the general formulas (1) and (2) are preferably salts of tetraalkylammonium, choline derivatives, or glycine derivatives, taking into account their availability and the impact on the biocompatibility of residual components when various forms are formed. Examples include halides, hydroxides, or carboxylates selected from tetraalkylammonium, choline, choline derivatives, glycine, and glycine derivatives, and further, tetraalkylammonium halides, as well as halides, hydroxides, or carboxylates selected from choline, choline derivatives, glycine, and glycine derivatives.

[0028] Specific examples of the above compounds include tetramethylammonium chloride, tetramethylammonium acetate, tetraethylammonium bromide, tetraethylammonium hydroxide, tetrapropylammonium chloride, tetrabutylammonium chloride, triethylmethylammonium chloride, trimethylphenylammonium chloride, choline, choline chloride, choline bitartrate, trimethylamine hydrochloride, triethanolamine hydrochloride, dibutylamine hydrochloride, glycine ethyl ester hydrochloride, glycinamide hydrochloride, methylpyridinium chloride, and 1,3-dimethylimidazolium chloride. More preferred specific examples include tetramethylammonium chloride, tetramethylammonium bromide, tetraethylammonium chloride, tetrapropylammonium chloride, tetrabutylammonium chloride, triethylmethylammonium chloride, choline chloride, choline bitartrate, glycine methyl ester hydrochloride, glycine ethyl ester hydrochloride, and glycinamide hydrochloride.

[0029] The amount of the compounds represented by general formulas (1) and (2) added to the fibroin aqueous solution of this embodiment is not particularly limited and can be adjusted as appropriate according to the desired properties. A suitable amount is 0.1% by mass or more and 50% by mass or less relative to the mass of fibroin dissolved in the aqueous solution. If the amount added is less than 0.1% by mass, sufficient storage stability cannot be obtained. On the other hand, if the amount added exceeds 50% by mass, high storage stability can be obtained, but it may become difficult to form into various forms.

[0030] The concentration of fibroin in the fibroin aqueous solution according to this embodiment is not particularly limited, but the effect is particularly high when the fibroin concentration is 5% by mass or more and 40% by mass or less of the total mass of the aqueous solution. Generally, the higher the fibroin concentration, the lower the storage stability of the aqueous solution tends to be, but when the fibroin concentration is less than 5% by mass, sufficient storage stability is often observed even without adding the additive of the present invention. On the other hand, when the fibroin concentration exceeds 40% by mass, it becomes difficult to prepare the aqueous solution itself, and sufficient storage stability may not be obtained even with the addition of the additive of the present invention.

[0031] The fibroin aqueous solution according to this embodiment can be produced by a scouring step of removing sericin from a fibroin raw material, a neutral salt dissolution step of dissolving the scouring fibroin raw material in a neutral salt aqueous solution to obtain a fibroin-neutral salt aqueous solution, a desalting step of desalting the fibroin-neutral salt aqueous solution to obtain a fibroin aqueous solution, an additive addition step of adding a compound represented by the general formula (1) or (2), and a concentration adjustment step of adjusting the concentration of the aqueous solution.

[0032] The scouring process, the neutral salt dissolution process, and the desalting process can be carried out using known methods and are not particularly limited.

[0033] The additive addition step can be performed at any of the above manufacturing steps, but it is preferable to perform it after the desalting step or the concentration adjustment step in order to control the concentration of the additive. The method of adding the additive can be appropriately selected from either directly adding the additive to the fibroin aqueous solution or dissolving the additive in water and then adding it as an aqueous solution, but adding it as an aqueous solution is preferable from the viewpoint of eliminating unevenness in the additive concentration. After adding the additive, it is preferable to stir the solution to make the additive concentration uniform. Since the fibroin aqueous solution may gel if subjected to strong shear force, it is necessary to select a stirring method that uses weak shear force.

[0034] The concentration adjustment step is preferably performed after the desalting step or after the additive addition step. In the concentration adjustment step, the fibroin aqueous solution can be diluted or concentrated to achieve the desired fibroin concentration.

[0035] When diluting an aqueous solution, it is preferable to add water to reach the desired concentration and then stir the solution until the entire solution has a uniform concentration. The stirring method is not particularly limited, but for the same reasons as above, a stirring method with weak shear force is preferred.

[0036] When concentrating an aqueous solution, known concentration methods can be used. The concentration method is not particularly limited, but methods that do not involve heat or shear force are preferred in order to suppress deterioration or denaturation of the fibroin aqueous solution. For example, methods such as ultrafiltration using a semipermeable membrane, dialysis, centrifugal concentration, and concentration under low temperature and reduced pressure are particularly preferred.

[0037] The production of fibroin aqueous solutions may further include a step to remove insoluble matter generated in the aqueous solution in order to further improve storage stability. By removing insoluble matter from the aqueous solution, gel formation can be suppressed, and the storage stability of the aqueous solution can be further improved.

[0038] Preferably, the process for removing insoluble matter includes a heating step of heating the fibroin aqueous solution at 65°C to 110°C for 30 minutes to 60 minutes, a cooling step of rapidly cooling the fibroin aqueous solution heated in the heating step to 15°C or below, and a microfiltration step of filtering the fibroin aqueous solution cooled in the cooling step using a microfiltration membrane.

[0039] In the heating step, the fibroin aqueous solution obtained in the desalting step described above is heated at a temperature of 65°C to 110°C for a period of 30 minutes to 60 minutes.

[0040] If the heating temperature is below 65°C, components that tend to become insoluble and form aggregates during the cooling process described later may not precipitate sufficiently, and there is a risk that sufficient aggregates cannot be formed during the cooling process. Furthermore, if the heating temperature exceeds 110°C, thermal denaturation of fibroin may progress, potentially degrading the quality. The heating temperature is preferably 65°C to 105°C, more preferably 70°C to 105°C, and even more preferably 85°C to 95°C. Within this range, sufficient aggregates can be formed during the cooling process and sufficiently removed during the microfiltration process, resulting in even better storage stability of the resulting fibroin aqueous solution.

[0041] If the heating time is shorter than 30 minutes, components that tend to become insoluble and form aggregates during the cooling process described later may not precipitate sufficiently, and there is a risk that sufficient aggregates will not be formed during the cooling process. In addition, there is a risk that microorganisms such as bacteria, which can cause quality deterioration during long-term storage, will not be sufficiently sterilized. On the other hand, if the heating time is longer than 60 minutes, there is a risk that thermal denaturation and gelation of fibroin will progress, leading to quality deterioration. The heating time is preferably 40 minutes or more and 60 minutes or less, more preferably 45 minutes or more and 60 minutes or less. Within this range, sufficient aggregates will be formed during the cooling process and can be sufficiently removed during the microfiltration process, resulting in even better storage stability of the resulting fibroin aqueous solution.

[0042] The heating method is not particularly limited, and conventionally known heating methods can be used. Specifically, examples include autoclaves, heaters, and microwave ovens.

[0043] In the cooling process, the fibroin aqueous solution heated in the heating process described above is rapidly cooled to 15°C or below. The advantages of rapid cooling include increased production efficiency, as suspended aggregates in the liquid quickly aggregate upon cooling, thus speeding up the precipitation and separation process; and hygienic and quality advantages, as it minimizes the time the liquid remains in a temperature range suitable for bacterial growth, thereby suppressing bacterial proliferation.

[0044] The cooling temperature is not particularly limited as long as it is 15°C or lower and does not freeze, but is preferably 0°C to 10°C, and more preferably 3°C to 8°C. When the cooling temperature is within this range, insoluble components or components that easily become insoluble in the heated fibroin aqueous solution tend to form aggregates, thus making the separation and removal from the fibroin aqueous solution even more reliable.

[0045] In this invention, rapid cooling generally refers to cooling at a cooling rate of 0.2°C / second or higher. The cooling rate is preferably 0.3°C / second to 0.8°C / second, more preferably 0.4°C / second to 0.7°C / second, and even more preferably 0.5°C / second to 0.6°C / second.

[0046] When the cooling rate is within this range, insoluble components or components that easily become insoluble in the heated fibroin aqueous solution tend to form aggregates, thus making the separation and removal from the fibroin aqueous solution even more reliable.

[0047] The method of rapid cooling is not particularly limited, but examples include using cooling means such as cold water, ice, ice water, dry ice, dry ice + ethanol, or a rapid cooler. Of these, ice water is preferred because it is easy to handle.

[0048] In the microfiltration process, the fibroin aqueous solution cooled in the aforementioned cooling process is filtered using a microfiltration membrane. Examples of materials used for microfiltration membranes include cellulose acetate, aromatic polyamide, polyvinyl alcohol, polysulfone, polyvinylidene fluoride, polyethylene, polyacrylonitrile, ceramic, polypropylene, polycarbonate, and fluororesin.

[0049] The pore size of the microfiltration membrane is not particularly limited, but is preferably 0.40 μm to 1.2 μm, more preferably 0.45 μm to 1.0 μm, and even more preferably 0.60 μm to 1.0 μm. Within this range, aggregates in the fibroin aqueous solution can be removed more quickly and sufficiently. The shape of the microfiltration membrane is not particularly limited, but examples include flat membranes, tubular membranes, spiral membranes, and hollow fiber membranes. Among these, hollow fiber membranes are preferred because they have low energy costs, can be used at relatively low pressures, and have a low risk of denaturation of protein components in the liquid due to pressure.

[0050] The fibroin aqueous solution of this embodiment can be used to produce various foams such as gels, sponges, films, and nonwoven fabrics. Foams produced using the fibroin aqueous solution of this embodiment can be called articles composed of fibroin. By including the compound represented by the general formula (1) or (2), the foam can be formed more quickly than when additives such as urea or guanidine hydrochloride are used. The fibroin aqueous solution of this embodiment is more effective in foams prepared in aqueous solutions, such as gels and sponges. The methods for producing these are not particularly limited, and known methods can be used; any external stimulus that promotes the crystallization (β-sheet formation) of fibroin can be used. For example, gels can be produced by changing the pH with hydrochloric acid, by using chemical substances such as gelling accelerators, by using shear force by strong stirring, or by applying an electric field. For sponges, methods using pollogens such as sodium chloride or sugar, or by freeze-drying the aqueous solution and then annealing it with heat or a solvent can be used. Furthermore, methods such as spray drying or freeze-drying can be used to produce the powder, and methods such as casting can be used to produce the film.

[0051] The article according to this embodiment comprises fibroin and a compound represented by the above general formula (1) or (2). The article according to this embodiment may be a solid substance having the form of a powder, film, or sponge, or it may have the form of a gel. Alternatively, the article according to this embodiment may be a molded body formed by a mold. [Examples]

[0052] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples unless it exceeds the essence of the invention. Regarding the amounts of components, "parts" and "%" refer to mass unless otherwise specified.

[0053] <Method for measuring the concentration of fibroin aqueous solution> The concentration of the fibroin aqueous solutions prepared in the following examples and comparative examples was determined by placing 0.5 mL of the fibroin aqueous solution into a glass container with a tare weight and drying it in an oven adjusted to 60°C for at least 2 hours. The solid content concentration was then calculated from the change in weight before and after drying.

[0054] <Method for measuring molecular weight> The molecular weight of the fibroin aqueous solutions prepared in the following examples and comparative examples was measured using the Agilent 2100 Bioanalyzer electrophoresis system (manufactured by Agilent) under the following conditions. • Microchip, separation matrix, fluorescent dye, electrophoresis buffer, molecular weight standard ladder: Agilent Protein 230 Kit • Control sample: Lyophilized bovine serum albumin powder, >96% (agarose gel electrophoresis) (Sigma-Aldrich, molecular weight 66.5 kDa) • Dilution and concentration of the silk fibroin aqueous solution and control sample: Using an 8M urea aqueous solution, the silk fibroin aqueous solution was diluted to 1.0-1.5% by mass / vol, and the control sample was diluted to approximately 1.3% by mass / vol. • Excitation wavelength: 630nm • Detection wavelength: 680nm The molecular weight of silk fibroin was calculated using the dedicated 2100 Expert software. The molecular weight of silk fibroin was calculated using a molecular weight calibration curve obtained from data of a molecular weight standard ladder measured together with the sample. The electrophoretic band used for molecular weight calculation was the band with the darkest color.

[0055] <Method for evaluating storage stability> Five mL of fibroin aqueous solutions prepared in the following examples and comparative examples were sealed in glass vials, and their storage stability was evaluated in a refrigerator at 4°C. The evaluation was performed visually every day, and the period during which the fibroin aqueous solution formed a gel and lost its fluidity was defined as the period during which it could be stored. The evaluation was performed according to the following criteria for aqueous solutions of the same molecular weight and concentration that did not contain additives (Comparative Examples 1-5). A: It can be stored for more than 30 days longer than additive-free products. B: It can be stored for 15 to 29 days longer than additive-free products. C: Can be stored for 5 to 14 days longer than additive-free products. D: Can be stored for up to 4 days, equivalent to additive-free products. E: Has a shorter shelf life than additive-free products. If the evaluation rank is A or B, the storage stability is considered good.

[0056] <Method for evaluating foam-forming properties> The foam-forming properties were evaluated by gel formation caused by pH changes using hydrochloric acid. A 9 mL fibroin aqueous solution was placed in a glass vial, and then 1 mL of 0.3 M hydrochloric acid was added dropwise. The container was gently shaken, and the mixture was left to stand in a 37°C incubator. The time until gel formation was measured. The evaluation was performed against aqueous solutions of the same molecular weight and concentration (Comparative Examples 1-5) that did not contain additives, according to the following criteria. A: It can form foam in the same amount of time as additive-free products. B: It takes 12 hours to 1 day longer to form foam compared to additive-free products. C: It takes 2 to 3 days longer to form foam compared to additive-free products. D: It takes more than 4 days longer to form the foam compared to additive-free products. If the evaluation rank was A or B, we judged that the foam-forming ability was good.

[0057] [Example 1] (scouring process) In a 5L glass beaker, 4.5L of ultrapure water was heated and brought to a boil. Then, 8.48g of sodium carbonate (manufactured by Kishida Chemical Co., Ltd.) was added to prepare a 0.02 mol / L sodium carbonate solution. Fibroin was obtained by adding 10g of cut silkworm cocoons (manufactured by Tajima Shoji Co., Ltd.), cut into approximately 1cm squares, and heating for 30 minutes to remove sericin. The fibroin was washed with cold ultrapure water, drained, and dried overnight in a fume hood to obtain refined fibroin.

[0058] (Neutral salt dissolution process) 80.7 g of anhydrous lithium bromide (manufactured by Kishida Chemical Co., Ltd.) was added to a volumetric flask and diluted to 100 mL to obtain a 9.3 mol / L lithium bromide aqueous solution. 3.0 g of refined fibroin was packed into a 100 mL glass beaker, and 14.8 mL of the 9.3 mol / L LiBr solution was added so that the refined fibroin was completely submerged. The solution was dissolved in a 60°C oven for 2 hours to obtain a clear neutral salt aqueous solution.

[0059] (Desalination process) 19 mL of the neutral salt aqueous solution prepared above was injected using a syringe into a dialysis cassette (Thermo Scientific) with a molecular weight cutoff of 3500 and a capacity of 30 mL. Dialysis was then performed by immersing the cassette in 2 L of ultrapure water. The water was changed 1 hour after the start of dialysis and again 4 hours later, and then once every 8 hours thereafter, for a total of 53 hours of dialysis to remove salt. The obtained aqueous solution was subjected to centrifugation twice at 11000 rpm, 4°C, and 20 minutes using a CR7N centrifuge (Eppendorf Highmac Technologies) to precipitate insoluble matter and obtain an aqueous fibroin solution. The solid content concentration of the obtained aqueous fibroin solution was 8%, and the molecular weight was 150 kDa.

[0060] (Additive addition process, concentration adjustment process) To 10 g of the above fibroin aqueous solution, 0.85 g of 7% tetramethylammonium chloride aqueous solution (7.4% relative to fibroin) and 0.58 g of ultrapure water were added, and the mixture was stirred using a mixing rotor until homogeneous to obtain the aqueous solution of Example 1 with a fibroin concentration of 7%. A storage stability test of the aqueous solution from Example 1 showed that it could be stored for 82 days. Furthermore, when the foam-forming properties were evaluated, a gel was formed in 6 hours.

[0061] [Examples 2-11, 16-23] Aqueous solutions for Examples 2-11 and 16-23 were prepared in the same manner as in Example 1, except that the compounds listed in Table 1 were added instead of tetramethylammonium chloride in the additive addition step. The concentration and amount of the additive aqueous solution, as well as the amount of ultrapure water added, were adjusted as appropriate to match the fibroin concentration and additive amount listed in Table 1.

[0062] [Example 12] The aqueous solution of Example 12 was prepared in the same manner as in Example 11, except that the heating time in the scouring process was changed from 30 minutes to 10 minutes.

[0063] [Example 13] The aqueous solution of Example 13 was prepared in the same manner as in Example 11, except that the heating time in the scouring process was changed from 30 minutes to 120 minutes.

[0064] [Examples 14, 15] After the desalting step, aqueous solutions for Examples 14 and 15 were prepared in the same manner as in Example 11, except that the aqueous solution concentrated in the following concentration step was used, and the fibroin concentration was adjusted to 15% or 25% in the concentration adjustment step.

[0065] (concentration process) A fibroin aqueous solution (solid content concentration 8%) after the desalination process was sealed in a dialysis tube (manufactured by Repligen) with a fractionation molecular weight cutoff of 10,000, and concentrated for 10 hours under conditions of 10°C and 5% RH while applying air from a blower. The solid content concentration of the resulting fibroin aqueous solution was 28%.

[0066] [Comparative Examples 1-5] Aqueous solutions of Comparative Examples 1 to 5 were prepared in the same manner as in Examples 1 and 12 to 15, except that no additives were added in the additive addition step.

[0067] [Comparative Examples 6-8] Aqueous solutions of Comparative Examples 6-8 were prepared in the same manner as in Example 1, except that the compounds listed in Table 1 were added instead of tetramethylammonium chloride in the additive addition step. The evaluation results of the fibroin aqueous solutions prepared as described above are summarized in Table 1.

[0068] [Table 1]

[0069] [Table 2]

[0070] As shown in Table 1, it can be seen that by including the compound represented by the general formula (1) or (2), an aqueous solution with excellent storage stability and foam-forming properties is obtained.

[0071] [Example 24] (Method for producing aqueous fibroin solution, including a step for removing insoluble matter) The aqueous solution of Example 24 was prepared in the same manner as in Example 1, except that after the desalting step of Example 1, a heating step, a cooling step, and a microfiltration step were performed.

[0072] (Heating, cooling, and microfiltration processes) The fibroin aqueous solution obtained in the desalination process of Example 1 was heated at 90°C for 45 minutes, and then rapidly cooled to 5°C using ice water. The resulting aqueous solution was microfiltered using a membrane filter (pore size 1 μm, hydrophilic PTFE, manufactured by Merck). The fibroin aqueous solution obtained above was subjected to the additive addition step and concentration adjustment step in the same manner as in Example 1 to obtain the fibroin aqueous solution of Example 24. Storage stability tests were conducted on the aqueous solutions of Example 24, and they were found to be able to be stored for more than 200 days. Furthermore, when foam formation was evaluated, gel formation occurred in 6 hours, similar to Example 1 and Comparative Example 1.

[0073] <Evaluation of foam formation properties by sponge formation> The foam-forming properties of the fibroin aqueous solutions of Example 1, Comparative Example 1, and Comparative Example 6 were evaluated by sponge formation.

[0074] [Example 25] 2 mL of the fibroin aqueous solution from Example 1 was placed in a plastic vial, and 4 g of sodium chloride, sieved to a particle size of 500-750 μm, was slowly added. The container was gently tapped to remove air bubbles, and then left in a 37°C incubator for 2 days. The resulting solid was left to stand in 1 L of ultrapure water for half a day to remove the sodium chloride particles. After repeating this 5 times, the resulting porous material was air-dried to obtain a fibroin sponge.

[0075] [Comparative Example 9] When a fibroin sponge was formed in the same manner as in Example 25, except that the fibroin aqueous solution of Comparative Example 1 was used, a fibroin sponge equivalent to that of Example 25 was obtained.

[0076] [Comparative Example 10] A fibroin sponge was formed using the same method as in Example 25, except that the fibroin aqueous solution of Comparative Example 6 was used. However, even after being left in an incubator for 10 days, no fibroin sponge was obtained.

[0077] <Evaluation of foam formation properties by gel formation using ultrasound> The foam-forming properties of the fibroin aqueous solutions from Example 1, Comparative Example 1, and Comparative Example 6 were evaluated by gel formation using ultrasound.

[0078] [Example 26] 5 mL of the fibroin aqueous solution from Example 1 was placed in a 15 mL conical tube, and ultrasonic irradiation was performed for 1 minute using an ultrasonic homogenizer (manufactured by Tommy Industries Co., Ltd.). The aqueous solution was left to stand in a 37°C incubator, and the time until gel formation was observed. A fibroin gel was obtained after 6 hours.

[0079] [Comparative Example 11] When a fibroin gel was formed in the same manner as in Example 26, except that the fibroin aqueous solution of Comparative Example 1 was used, a fibroin gel was obtained after 6 hours, similar to Example 26.

[0080] [Comparative Example 12] When a fibroin gel was formed using the same method as in Example 26, except that the fibroin aqueous solution of Comparative Example 6 was used, it took 7 days to obtain the gel. [Industrial applicability]

[0081] The present invention provides a fibroin aqueous solution with excellent storage stability and foam-forming properties, as well as a method for producing the same.

[0082] Embodiments of the present invention include the following configurations and methods. [Configuration 1] An aqueous fibroin solution containing fibroin and a compound represented by the following general formula (1) or (2). [ka] [In general formula (1), R 1 From R 4 Each of them operates independently. hydrogen atom, An aliphatic hydrocarbon group having 1 to 8 carbon atoms, which may have substituents, or This represents an aryl group or aralkyl group having 6 to 10 carbon atoms, which may have substituents. However, R 1 From R 4 At least one of them is not a hydrogen atom, X - This indicates an anion. [ka] [In general formula (2), CN=C and A both form a ring structure. R 5 teeth, hydrogen atom, An aliphatic hydrocarbon group having 1 to 8 carbon atoms, which may have substituents, or This represents an aryl group or aralkyl group having 6 to 10 carbon atoms, which may have substituents. X - This indicates an anion. [Configuration 2] It contains the compound represented by the general formula (1) above, In the above general formula (1), R 1 From R 4 Each of them operates independently. hydrogen atom, An alkyl group having 1 to 8 carbon atoms may have a hydroxyl group, an amino group, an alkoxycarbonyl group, or a carbamoyl group as a substituent, or Representing an aryl group or aralkyl group with 6 to 10 carbon atoms, A fibroin aqueous solution as described in Composition 1. [Configuration 3] It contains the compound represented by the general formula (2) above, In the above general formula (2), CN=C and A both constitute pyridine or imidazole. R 5 teeth, hydrogen atom, Alkyl groups having 1 to 8 carbon atoms, Representing an aryl group or aralkyl group with 6 to 10 carbon atoms, A fibroin aqueous solution as described in Composition 1. [Structure 4] In the above general formula (1) or the above general formula (2), X - It is selected from halide ions, hydroxide ions, and carboxylate anions. A fibroin aqueous solution as described in any one of items 1 to 3. [Composition 5] The compound contains the compound represented by the general formula (1), and the compound represented by the general formula (1) is An aqueous fibroin solution according to composition 1, wherein the halogenated compound, hydroxide, or carboxylate is selected from tetraalkylammonium, choline, choline derivatives, glycine, and glycine derivatives. [Composition 6] The compound contains the compound represented by the general formula (1), and the compound represented by the general formula (1) is An aqueous fibroin solution according to configuration 1, which is a tetraalkylammonium halide, and any halide, hydroxide, or carboxylate selected from choline, choline derivatives, glycine, and glycine derivatives. [Composition 7] An aqueous fibroin solution according to any one of the constructs 1 to 6, wherein the molecular weight of the fibroin is 100,000 or more. [Structure 8] An aqueous fibroin solution according to any one of the configurations 1 to 7, wherein the concentration of the fibroin is 5% by mass or more and 40% by mass or less. [Composition 9] The fibroin aqueous solution described in any one of the constructs 1 to 8, derived from silk. [Method 1] The process involves adding a compound represented by the following general formula (1) or (2) to an aqueous fibroin solution, A method for producing an aqueous fibroin solution, comprising a step of adjusting the concentration of the obtained solution. [ka] [In general formula (1), R 1 From R 4 Each of them operates independently. hydrogen atom, An aliphatic hydrocarbon group having 1 to 8 carbon atoms, which may have substituents, or This represents an aryl group or aralkyl group having 6 to 10 carbon atoms, which may have substituents. However, R 1 From R 4 At least one of them is not a hydrogen atom, X - This indicates an anion. [ka] [In general formula (2), CN=C and A both form a ring structure. R 5 teeth, hydrogen atom, An aliphatic hydrocarbon group having 1 to 8 carbon atoms, which may have substituents, or This represents an aryl group or aralkyl group having 6 to 10 carbon atoms, which may have substituents. X - This indicates an anion. [Method 12] moreover, A neutral salt dissolution step is performed by dissolving the refined fibroin raw material in a neutral salt aqueous solution to obtain a fibroin-neutral salt aqueous solution, A desalting step is performed to desalt the fibroin-neutral salt aqueous solution to obtain a fibroin aqueous solution, A heating step of heating the fibroin aqueous solution at 65°C to 110°C for a period of 30 minutes to 60 minutes, A cooling step in which the fibroin aqueous solution heated in the above heating step is rapidly cooled to 15°C or below, A microfiltration step is performed in which the fibroin aqueous solution cooled in the above cooling step is filtered using a microfiltration membrane. including, A method for producing an aqueous fibroin solution as described in Method 1. [Configuration 10] An article comprising fibroin and a compound represented by the following general formula (1) or (2). [ka] [In general formula (1), R 1 From R 4 Each of them operates independently. hydrogen atom, An aliphatic hydrocarbon group having 1 to 8 carbon atoms, which may have substituents, or This represents an aryl group or aralkyl group having 6 to 10 carbon atoms, which may have substituents. However, R 1 From R 4 At least one of them is not a hydrogen atom, X - This indicates an anion. [ka] [In general formula (2), CN=C and A both form a ring structure. R 5 teeth, hydrogen atom, An aliphatic hydrocarbon group having 1 to 8 carbon atoms, which may have substituents, or This represents an aryl group or aralkyl group having 6 to 10 carbon atoms, which may have substituents. X - This indicates an anion. [Composition 11] The article according to configuration 10, which is in the form of a powder, film, sponge, or gel, or is a molded body formed by a mold. [Composition 12] The article according to composition 11 or 12, wherein the fibroin is derived from silk.

Claims

1. An aqueous fibroin solution containing fibroin and a compound represented by the following general formula (1). 【Chemistry 1】 [In general formula (1), R １ From R ４ Each of them operates independently. hydrogen atom, An aliphatic hydrocarbon group having 1 to 8 carbon atoms, which may have a hydroxyl group, an amino group, an alkoxycarbonyl group, or a carbamoyl group as a substituent, or It represents an aryl group or aralkyl group having 6 to 10 carbon atoms, However, R １ From R ４ At least one of them is not a hydrogen atom, X － This indicates an anion.

2. In the above general formula (1), X － It is selected from halide ions, hydroxide ions, and carboxylate anions. The aqueous fibroin solution according to claim 1.

3. The compound represented by the general formula (1) is The aqueous fibroin solution according to claim 1, which is a halogenated compound, hydroxide, or carboxylate selected from tetraalkylammonium, choline, choline derivatives, glycine, and glycine derivatives.

4. The compound represented by the general formula (1) is An aqueous fibroin solution according to claim 1, wherein the tetraalkylammonium halide is selected from choline, choline derivatives, glycine, and glycine derivatives, and is selected from a halide, hydroxide, or carboxylate.

5. The aqueous fibroin solution according to any one of claims 1 to 4, wherein the concentration of the fibroin is 5% by mass or more and 40% by mass or less.

6. The aqueous fibroin solution according to any one of claims 1 to 4, wherein the fibroin is derived from silk.

7. The aqueous fibroin solution according to any one of claims 1 to 4, wherein the compound represented by the general formula (1) is soluble in water at a concentration of 0.01% by mass or more.

8. The aqueous fibroin solution according to any one of claims 1 to 4, wherein the compound represented by the general formula (1) is contained in an amount of 0.1% by mass or more and 50% by mass or less based on the mass of the fibroin.

9. The aqueous fibroin solution according to any one of claims 1 to 4, wherein the molecular weight of the fibroin is 100,000 or more and 350,000 or less.

10. The process involves adding a compound represented by the following general formula (1) to an aqueous fibroin solution, A method for producing an aqueous fibroin solution, comprising a step of adjusting the concentration of the obtained solution. 【Chemistry 2】 [In general formula (1), R １ From R ４ Each of them operates independently. hydrogen atom, An aliphatic hydrocarbon group having 1 to 8 carbon atoms, which may have a hydroxyl group, an amino group, an alkoxycarbonyl group, or a carbamoyl group as a substituent, or It represents an aryl group or aralkyl group having 6 to 10 carbon atoms, However, at least one of R １ from R ４ is not a hydrogen atom, X － This indicates an anion.

11. moreover, A neutral salt dissolution step is performed by dissolving the refined fibroin raw material in a neutral salt aqueous solution to obtain a fibroin-neutral salt aqueous solution, A desalting step is performed to desalt the fibroin-neutral salt aqueous solution to obtain a fibroin aqueous solution, A heating step of heating the fibroin aqueous solution at 65°C to 110°C for a period of 30 minutes to 60 minutes, A cooling step is performed to rapidly cool the fibroin aqueous solution heated in the heating step down to 15°C or below. A microfiltration step is performed in which the fibroin aqueous solution cooled in the above cooling step is filtered using a microfiltration membrane. including, A method for producing an aqueous fibroin solution according to claim 10.

12. The method for producing an aqueous fibroin solution according to claim 11, further comprising an external stimulation step to promote the crystallization of the fibroin in the aqueous fibroin solution.

13. An article comprising fibroin and a compound represented by the following general formula (1). 【Transformation 3】 [In general formula (1), R １ From R ４ Each of them operates independently. hydrogen atom, An aliphatic hydrocarbon group having 1 to 8 carbon atoms, which may have a hydroxyl group, an amino group, an alkoxycarbonyl group, or a carbamoyl group as a substituent, or It represents an aryl group or aralkyl group having 6 to 10 carbon atoms, However, R １ From R ４ At least one of them is not a hydrogen atom, X － This indicates an anion.

14. The article according to claim 13, having the form of a powder, film, sponge, or gel, or being a molded body formed by a mold.

15. The article according to claim 13 or 14, wherein the fibroin is derived from silk.

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